Seven nptII and gus transgenic lines of the apple (Malus ×domestica Borkh.) rootstock Malling 7 (M.7) were examined by glucuronidase (GUS) histochemical testing and a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). These lines had different amounts of neomycin phosphotransferase II (NPTII). The amounts of NPTII among lines was positively correlated with the ability of the transgenic lines to regenerate in the presence of kanamycin, paromomycin, or geneticin. Regenerants derived from transgenic lines also varied greatly in GUS expression. The apical portion of regenerant stem tissues had stronger GUS staining than the basal portion of stem. All regenerated tissue of T1, a transgenic line originally classified as a uniform GUS staining line, showed non-GUS staining, while the regenerated tissues of chimeric transgenic lines showed nonstaining, chimeric staining, or uniform GUS staining, indicating the potential to select uniform GUS staining lines from chimeras. Polymerase chain reaction (PCR) indicated the gus gene was present in GUS negative (nonstaining) lines. Negative PCR results with primers derived from vir G of Agrobacterium tumefaciens, and failure to isolate A. tumefaciens from M.7 transgenics indicated that PCR and GUS staining results were not due to A. tumefaciens. A modified PCR methylation assay (MPMA) indicated that methylation of cytosines of the CCGG site in the gus gene, and in the border between the CaMV35S promoter and the gus gene, was positively correlated with complete gus gene silencing (nonstaining lines). However, the MPMA indicated that methylation was not always associated with variable GUS expression, suggesting that chimeric staining could be due to a mixture of transformed and nontransformed cells in some transgenic lines.
Kisung Ko, Susan K. Brown, John L. Norelli, and Herb S. Aldwinckle
Kisung Ko, John L. Norelli, Jean-Paul Reynoird, Herb S. Aldwinckle, and Susan K. Brown
Genes encoding lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of `Galaxy' apple, to enhance resistance to Erwinia amylovora. In these plasmids, the T4L gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some T4L or attE transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between attE and T4L with respect to resistance to E. amylovora.